Tumor suppressor p53 is known to be a direct transcriptional target of type I interferons (IFNs), contributing to virus-induced apoptosis, and in turn activating itself the interferon pathway. Acetylation, among many other post-translational modifications of p53, is thought to exert a crucial role regulating p53 activity. Here, we examined the contribution of this modification on the antiviral activity mediated by p53. Our results show that virus infection induces p53 acetylation at lysine 379, and that this modification is absolutely required for p53-dependent transcriptional transactivation of both, pro-apoptotic and IFN-stimulated genes induced by virus infection, and for p53-mediated control of virus replication. Thus, our study identifies p53 acetylation as an indispensable event that enables the p53-mediated antiviral response.